Treatment of chronic pelvic pain syndrome

ABSTRACT

The present invention provides compositions and methods for detection, diagnosis, treatment and/or prevention of chronic pelvic pain syndrome. In particular, the present invention provides biomarkers of chronic pelvic pain syndrome (e.g., mast cell markers (e.g., tryptase)), and/or inhibition of mast cell function (e.g. inhibition of MCP-1 and/or MIP-1α) to treat or prevent chronic pelvic pain syndrome.

This application is a continuation of U.S. patent application Ser. No.13/118,156, filed May 27, 2011, which claims priority to U.S.Provisional Patent Application Ser. No. 61/349,282, filed May 28, 2010,and U.S. Provisional Patent Application Ser. No. 61/349,277, filed May28, 2010, each of which are herein incorporated by reference in theirentireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No.1K01DK079019-01A2 awarded by the National Institutes of Health, NationalInstitute of Diabetes and Digestive and Kidney Diseases. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention provides compositions and methods for detection,diagnosis, treatment and/or prevention of chronic pelvic pain syndrome.In particular, the present invention provides biomarkers of chronicpelvic pain syndrome (e.g., mast cell markers (e.g., tryptase)), and/orinhibition of mast cell function (e.g. inhibition of MCP-1 and/orMIP-1α) to treat or prevent chronic pelvic pain syndrome.

BACKGROUND OF THE INVENTION

Chronic prostatitis/chronic pelvic pain syndrome (collectively referredto herein as CPPS) is a syndrome of undetermined etiology occurring inmen. CPPS is the third of four subgroups of prostatitis recognized bythe National Institutes of Health. Category I encompasses acutebacterial prostatitis, and Category II covers chronic bacterialinfection. Category III, CPPS, includes all remaining prostatitissyndromes, and is subdivided into IIIa (inflammatory) and IIIb(non-inflammatory). These sub-categories can be distinguished by thepresence of leukocytosis in expressed prostatic secretions or sedimentin a post-massage urine sample. Category IV represents asymptomaticprostatitis, which often is associated with benign prostate hyperplasia.

Prostatitis is extraordinarily common, resulting in approximately 2million office visits to primary care physicians and urologists in theUnited States annually (1997 American Urological Association AnnualMeeting, National Ambulatory Medical Care Survey, National Center forHealth Statistics, 1990 to 1994). Patients with CPPS suffer fromchronic, episodic pain in the perineum or pelvic region, irritative andobstructive voiding symptoms, and adverse effects upon sexual function(Alexander et al., Urology 48:568-574 (1996)). Men with chronicprostatitis often require repeated physician visits. Medicalexpenditures relating to CPPD are conservatively estimated to exceedhalf a billion dollars annually.

While an enormous number of patients seek the care of a physicianbecause of prostatitis-like symptoms, almost nothing is known aboutdiagnostic criteria, etiology, or objective signs for CPPS. Pain in thepelvic region is the most frequently reported and the most severesymptom in patients with CPPS (Alexander et al., Urology 48:568-74(1996)). It was because of these observations and the paucity ofobjective criteria for defining the disease, which the NationalInstitute of Diabetes and Digestive and Kidney Diseases working group inprostatitis suggested that the disease be named Chronic Pelvic PainSyndrome.

One reason for the present state of confusion regarding CPPS is thesimilarity of CPPS symptoms to the symptoms of bacterial prostatitis.Only about 5 to 10% of patients whose symptoms are consistent withbacterial prostatitis are shown to have infection in the prostate gland(Weidner et al., Infection 19:S109-S190 (1991)). The misdiagnosis ofCPPS as infectious prostatitis commonly results in unnecessary treatmentwith multiple courses of antibiotics at burdensome costs to patients andto the health care system with no demonstrated benefit to patients.

Much effort has been expended to identify an organism underlying thecause of CPPS but no clear consensus has emerged identifying any suchorganism as the causative agent. Additionally, some men with CPPS haveevidence of inflammation of the prostate. While the cellular andcytokine mediators involved in the inflammatory process have beenincreasingly clarified in the immunologic literature, few studies haveinvestigated the immunobiology of the prostate gland to determinewhether CPPS might arise from an auto-immune-like condition.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides compositions andmethods for treatment and/or prevention of chronic pelvic pain syndrome.In particular, the present invention provides compositions and methodsfor inhibition of mast cell function to treat or prevent chronic pelvicpain syndrome. In some embodiments, chronic pelvic pain syndrome istreated or prevented by inhibition of mast cell function and one or moreadditional therapies (e.g. inhibition of MCP-1 and MIP-1α).

In some embodiments, the present invention provides a method fortreating and/or preventing chronic pelvic pain syndrome in a subject,comprising administering therapeutic composition comprising atherapeutically effective amount of an inhibitor of mast cell functionto the subject. In some embodiments, the therapeutic composition furthercomprises an inhibitor of MCP-1 and/or MIP-1α. In some embodiments, thetherapeutic composition comprises two or more inhibitors of MCP-1 and/orMIP-1α. In some embodiments, the inhibitor of MCP-1 and/or MIP-1αcomprises an antisense oligonucleotide, genetic therapy, oranti-chemokine therapy. In some embodiments, the inhibitor of mast cellfunction comprises a mast cell stabilizer and/or histamine receptorantagonist. In some embodiments, the mast cell stabilizer comprisessodium, lodoxamide, nedocromil, and/or derivatives thereof. In someembodiments, the histamine receptor antagonist comprises a histaminereceptor 1 antagonist. In some embodiments, the histamine receptor 1antagonist comprises: clemastine, diphenhydramine, doxylamine,loratadine, desloratadine, fexofenadine, pheniramine, cetirizine,ebastine, promethazine, chlorpheniramine, levocetirizine, quetiapine,meclizine, dimenhydrinate, and/or derivatives thereof. In someembodiments, the histamine receptor antagonist comprises a histaminereceptor 2 antagonist. In some embodiments, the histamine receptor 1antagonist comprises: cimetidine, famotidine, ranitidine, nizatidine,roxatidine, lafutidine, and derivatives thereof.

In some embodiments, the present invention provides a method fortreating and/or preventing chronic pelvic pain syndrome in a subject,comprising co-administering an inhibitor of mast cell function, and oneor more inhibitors of MCP-1 or MIP-1α. In some embodiments, the one ormore inhibitors of MCP-1 or MIP-1α comprise inhibitors of MCP-1 andMIP-1α. In some embodiments, the one or more inhibitors of MCP-1 orMIP-1α comprise an antisense oligonucleotide, genetic therapy, oranti-chemokine therapy. In some embodiments, the inhibitor of mast cellfunction comprises a mast cell stabilizer, inhibitor of degranulation,or histamine receptor antagonist. In some embodiments, the mast cellstabilizer comprises sodium, lodoxamide, nedocromil, and/or derivativesthereof. In some embodiments, the histamine receptor antagonistcomprises a histamine receptor 1 antagonist. In some embodiments, thehistamine receptor 1 antagonist comprises: clemastine, diphenhydramine,doxylamine, loratadine, desloratadine, fexofenadine, pheniramine,cetirizine, ebastine, promethazine, chlorpheniramine, levocetirizine,quetiapine, meclizine, dimenhydrinate, and/or derivatives thereof. Insome embodiments, the histamine receptor antagonist comprises ahistamine receptor 2 antagonist. In some embodiments, the histaminereceptor 1 antagonist comprises: cimetidine, famotidine, ranitidine,nizatidine, roxatidine, lafutidine, and derivatives thereof.

In some embodiments, the present invention provides a composition forthe treatment or prevention of CPPS, comprising: (a) an inhibitor ofmast cell function, and (b) an inhibitor of a CPPS biomarker. In someembodiments, the inhibitor of mast cell function comprises a mast cellstabilizer, inhibitor of degranulation, or histamine receptorantagonist. In some embodiments, the mast cell stabilizer comprisessodium, lodoxamide, nedocromil, and/or derivatives thereof. In someembodiments, the histamine receptor antagonist comprises a histaminereceptor 1 antagonist. In some embodiments, the histamine receptor 1antagonist comprises: clemastine, diphenhydramine, doxylamine,loratadine, desloratadine, fexofenadine, pheniramine, cetirizine,ebastine, promethazine, chlorpheniramine, levocetirizine, quetiapine,meclizine, dimenhydrinate, and/or derivatives thereof. In someembodiments, the present invention comprises two or more inhibitors ofbiomarkers of CPPS. In some embodiments, the biomarkers of CPPS areselected from MCP-1 and MIP-1α. In some embodiments, the biomarkers ofCPPS comprise inhibitors of MIP-1α and MCP-1.

In some embodiments, the present invention provides biomarkers ofchronic pelvic pain syndrome for use in diagnosis, drug screening,therapy monitoring, research and therapeutic applications. Inparticular, the present invention provides mast cell markers (e.g.,tryptase) as biomarkers of chronic pelvic pain syndrome.

Accordingly, in some embodiments, the present invention provides amethod for detecting chronic pelvic pain syndrome in a subject,comprising providing a sample from a subject; and detecting theexpression of a mast cell-specific marker (e.g., tryptase) in thesample. Any mast cell-specific marker or a combination of markersspecific for mast cells (a mast cell fingerprint) may be used. In someembodiments, one or more proteins, peptides, or nucleic acid molecules(e.g., DNA, RNA) is detected or quantified. Embodiments of the inventionare illustrated below using mast cell tryptase as an exemplary marker.It should be understood that in some embodiments, similar methods, kits,compositions, and the like may be employed with other mast-specificmarkers.

In some embodiments, detecting the expression of mast cell tryptasecomprises detecting the presence of mast cell tryptase mRNA. In someembodiments, detecting expression of mast cell tryptase comprisesexposing the mast cell tryptase mRNA to a nucleic acid probecomplementary to the mast cell tryptase mRNA. In some embodiments,detecting expression of mast cell tryptase comprises detecting thepresence of a mast cell tryptase polypeptide. In some embodiments,detecting the presence of a mast cell tryptase polypeptide comprisesexposing the mast cell tryptase polypeptide to an antibody specific tothe mast cell tryptase polypeptide and detecting the binding of theantibody to the mast cell tryptase polypeptide. In some embodiments, thesubject comprises a human subject. In some embodiments, the samplecomprises semen, seminal fluid, and/or expressed prostatic secretions.

The present invention also provides a method for selecting a therapeuticcourse of action, comprising providing a sample from a subject;detecting the expression of mast cell tryptase in the sample; andtreating the subject based upon the expression of mast cell tryptase.

The present invention provides a kit for characterizing CPPS in asubject, comprising a reagent capable of specifically detecting thepresence or absence of expression of mast cell tryptase; and,optionally, instructions for using the kit for characterizing pain inthe subject. In some embodiments, the reagent comprises a nucleic acidprobe complementary to a mast cell tryptase mRNA. In some embodiments,the reagent comprises an antibody that specifically binds to a mast celltryptase polypeptide.

The present invention also provides a method of screening compounds,comprising providing a sample; and one or more test compounds; andcontacting the sample with the test compound; and detecting a change inmast cell tryptase expression in the sample in the presence of the testcompound relative to the absence of the test compound. In someembodiments, the detecting comprises detecting mast cell tryptase mRNA.In some embodiments, the detecting comprises detecting mast celltryptase polypeptide. In some embodiments, the cell is in vitro or invivo. In some embodiments, the test compound comprises an antisensecompound. In some embodiments, the test compound comprises a smallmolecule drug.

In some embodiments, the present invention provides a method fordetecting chronic pelvic pain syndrome in a subject, comprisingproviding a sample from a subject; and detecting the expression of mastcell tryptase, along with one or more additional biomarkers of chronicpelvic pain syndrome, in the sample. In some embodiments, additionalbiomarkers of chronic pelvic pain syndrome comprise MIP-1α and/or MCP-1.

In some embodiments, the present invention provides a kit forcharacterizing CPPS in a subject, comprising a reagent capable ofspecifically detecting the presence or absence of expression of mastcell tryptase and one or more additional biomarkers of chronic pelvicpain syndrome. In some embodiments, additional biomarkers of chronicpelvic pain syndrome comprise MIP-1α and/or MCP-1.

The present invention also provides a method of screening compounds,comprising providing a sample; and one or more test compounds; andcontacting the sample with the test compound; and detecting a change inexpression of mast cell tryptase and one or more additional biomarkersof chronic pelvic pain syndrome in the sample in the presence of thetest compound relative to the absence of the test compound. In someembodiments, additional biomarkers of chronic pelvic pain syndromecomprise MIP-1α and/or MCP-1.

The present invention also provides a panel of markers for detection ofCPPS in a subject, comprising mast cell tryptase, and one or moreadditional markers of CPPS. In some embodiments, additional markers ofCPPS comprise MIP-1α and/or MCP-1.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the EAP induces chronic pelvic pain in male NOD mice. Micewere assessed for tactile allodynia with von Frey filaments before andafter immunization. Sham-injected male NOD mice received adjuvantinjection while experimental NOD mice were injected with antigen. ANOVAexhibited significantly increased responses at all filaments tested inPAg-treated mice at days 10-30, with no significant differences inbaseline between controls and PAg-treated mice. EAP induced 300-400%increase in pain compared to controls.

FIG. 2 shows EAP induces chronic pelvic pain in male B6 mice. Tactileallodynia was measured as responses to mechanical stimulation of thepelvic region and hind paw using von Frey filaments of 5 calibratedforces. A) Responses to pelvic stimulation of sham-injected male B6 micereceiving injection Titer Max. B) Responses to pelvic stimulation ofmale B6 mice injected with PAg. ANOVA indicated an increase in responsefrequency from baseline at all filaments tested in PAg-treated mice atdays 15-20 with no significant differences in baseline between controlsand PAg treated mice. C) PAg induced no significant change in tactilesensitivity (50% threshold) of the plantar region of the paw.

FIG. 3 shows mast cell recruitment and activation are observed in theprostates of EAP mice. NOD mouse prostate sections were stained with theacidified toluidine blue and total mast cells, resting mast cells, andactivated mast cells were quantified in a blinded fashion. Beforeinduction of EAP, few mast cells that were largely resting (A. seearrows and t=0) were observed. At 10, 20 and 30 days after inductionlarger number of mast cells of an activated phenotype were observed (seeT=10, 20 and 30). Total mast cell numbers were increased till 20 days(B), with significant majority of cells being of the resting phenotypeat 5 and 10 days (C). Activated cells were observed to be maximal at 10days followed by a reduction in detection.

FIG. 4 shows pelvic pain behavior in wild type (C57BLJ6J) and mast celldeficient mice.

FIG. 5 shows EAP in mast cell deficient (KitW^(−sh)/KitW^(−sh)) mice isassociated with chronic inflammation.

FIG. 6 shows Anti-MCP-1 or Anti-MIP-1α antibodies reduce paindevelopment in EAP.

FIG. 7 shows Anti-MCP-1 antibody therapy reduces pain in EAP.

FIG. 8 shows functional inhibitors of mast cell products or mast celldegranulation therapeutically reduce pelvic pain in EAP. Mice injectedwith rat prostate antigen (PAg) were tested at baseline and 10 daysafter injection for the development of pain behavior. Mice were treatedat day 10 by intraperitoneal injection with saline (Control, A), 10mg/kg each of cetrizine (H1, B), ranitidine (H2, C), Cromolyn sodium (MCstabilizer, D) or all three drugs (Combination, E) in a 200 μl volume.Following blinding of treatment and control groups, pelvic pain behaviorwas tested one hour after drug administration.

FIG. 9 shows mast cell tryptase was significantly elevated in EPS fromCPPS but not in BPH. Samples were normalized (200 ug) for proteinconcentration and tryptase activity was calculated relative to apositive standard.

FIG. 10 shows an exemplary experimental design to assess the efficacy ofmast cell inhibitors on chronic pelvic pain. EAP induction occurred for20 days in NOD mice. Pain was assessed at the baseline, 20 days, 1 hourpost treatment, 24 hours post treatment, and 1 hour post secondtreatment.

FIG. 11 shows a chart of treatment groups assessed. One group was givenoral administered cromolyn sodium at 0.5 mg/kg. A second group was givenintraperitoneal administered cromolyn sodium at 1.0 mg/kg. Twoadditional groups were given intravenous administered cromolyn sodium at0.5 mg/kg in combination with Cetirizine (H1) 2.5 mg/kg, or cromolynsodium at 1.0 mg/kg in combination with Cetirizine (H1) 2.5 mg/kg.

FIG. 12 shows histograms demonstrating the decrease in pain resultingfrom oral administration at the 24 hour time point and the 1 hour timepoint after the second treatment are the time points in whichsignificant pain reduction was found for the oral therapy. The absenceof a bar in panel B indicates a pain increase. Pain reduction wascompared to intraporstatic lidocaine which is current standard of careand with Cetirizine alone (dotted horizontal lines).

FIG. 13 shows a histogram demonstrating the decrease in pain resultingfrom intraperitoneal administration of cromolyn sodium alone or incombination with Cetirizine (H1) at the 24 hour time point. Painreduction was compared to intraporstatic lidocaine which is currentstandard of care and with Cetirizine alone (dotted horizontal lines).

FIG. 14 shows a histogram demonstrating the decrease in pain resultingfrom intravenous administration of cromolyn sodium alone or incombination with Cetirizine (H1) at the 24 hour time point. Painreduction was compared to intraporstatic lidocaine which is currentstandard of care and with Cetirizine alone (dotted horizontal lines).

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

The term “epitope” as used herein refers to that portion of an antigenthat makes contact with a particular antibody.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment or subject to various tests (e.g., diagnostic tests) that maybe provided by the present invention. Typically, the terms “subject” and“patient” are used interchangeably herein in reference to a humansubject.

As used herein, the term “subject suspected of having CPPS” refers to asubject that presents one or more symptoms indicative of chronic pelvicpain syndrome (CPPS) or is being screened for CPPS (e.g., during aroutine physical). A subject suspected of having CPPS may also have oneor more risk factors. A subject suspected of having CPPS has generallynot been tested for CPPS. However, a “subject suspected of having CPPS”encompasses an individual who has received an initial diagnosis but forwhom the nature of the CPPS is not known. The term further includespeople who once had CPPS.

As used herein, the term “subject at risk for CPPS” refers to a subjectwith one or more risk factors for developing CPPS.

As used herein, the term “characterizing CPPS in a subject” refers tothe identification of one or more properties of CPPS in a subject.

The terms “test compound” and “candidate compound” refer to any chemicalentity, pharmaceutical, drug, and the like that is a candidate for useto treat or prevent a disease, illness, sickness, or disorder of bodilyfunction (e.g., CPPS). Test compounds comprise both known and potentialtherapeutic compounds. A test compound can be determined to betherapeutic by screening using the screening methods of the presentinvention.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include, amongother things, body fluids (e.g., semen), blood products (e.g., plasma,serum and the like), and their component parts (e.g., expressedprostatic secretions, termed “ESPs” herein, seminal plasma or seminalfluid). Environmental samples include environmental material such assurface matter, soil, water, and industrial samples. Such examples arenot however to be construed as limiting the sample types applicable tothe present invention.

As used herein, the term “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments can consist of, but are not limitedto, test tubes and cell culture. The term “in vivo” refers to thenatural environment (e.g., an animal or a cell) and to processes orreaction that occur within a natural environment.

When a protein or fragment of a protein is used to immunize a hostanimal, numerous regions of the protein may induce the production ofantibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as “antigenic determinants”. An antigenic determinantmay compete with the intact antigen (i.e., the “immunogen” used toelicit the immune response) for binding to an antibody.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A,” the presence of aprotein containing epitope A (or free, unlabelled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

As used herein, the terms “non-specific binding” and “backgroundbinding” when used in reference to the interaction of an antibody and aprotein or peptide refer to an interaction that is not dependent on thepresence of a particular structure (i.e., the antibody is binding toproteins in general rather that a particular structure such as anepitope).

As used herein, the term “CPPS marker” or “CPPS marker genes” refers toa biomarker (e.g., gene) whose presence, absence, concentration, and/orexpression level (e.g., as detected by mRNA or protein expression),alone or in combination with other markers, is correlated with CPPS orprognosis of CPPS. The correlation may relate to either an increased ordecreased expression of the gene. For example, the expression of thegene may be indicative of CPPS, or reduced level of expression of thegene may be correlated with response to therapy for CPPS in a CPPSpatient.

As used herein, the term “a reagent that specifically detects expressionlevels” refers to reagents used to detect the expression of one or moregenes or proteins (e.g., including but not limited to, the CPPS markersof the present invention). Examples of suitable reagents include but arenot limited to, nucleic acid probes capable of specifically hybridizingto the gene of interest, PCR primers capable of specifically amplifyingthe gene of interest, and antibodies capable of specifically binding toproteins expressed by the gene of interest. Other non-limiting examplescan be found in the description and examples below.

As used herein, the term “detecting a decreased or increased expressionrelative to non-CPPS control” refers to measuring the level ofexpression of a gene (e.g., the level of mRNA or protein) relative tothe level in a non-CPPS control sample. Gene expression can be measuredusing any suitable method, including but not limited to, those describedherein.

As used herein, the term “detecting a change in gene expression (e.g.,mast cell tryptase) in a sample in the presence of said test compoundrelative to the absence of said test compound” refers to measuring analtered level of expression (e.g., increased or decreased) in thepresence of a test compound relative to the absence of the testcompound. Gene expression can be measured using any suitable method.

As used herein, the term “instructions for using said kit for detectingCPPS in said subject” includes instructions for using the reagentscontained in the kit for the detection and characterization of CPPS in asample from a subject.

As used herein, the term “CPPS expression profile map” refers to apresentation of expression levels of genes in a sample (e.g., prostatetissue or seminal fluid) The map may be presented as a graphicalrepresentation (e.g., on paper or on a computer screen), a physicalrepresentation (e.g., a gel or array) or a digital representation storedin computer memory. Each map corresponds to a particular type of sampleand thus provides a template for comparison to a patient sample.

As used herein, the terms “computer memory” and “computer memory device”refer to any storage media readable by a computer processor. Examples ofcomputer memory include, but are not limited to, RAM, ROM, computerchips, digital video disc (DVDs), compact discs (CDs), hard disk drives(HDD), and magnetic tape.

As used herein, the term “computer readable medium” refers to any deviceor system for storing and providing information (e.g., data andinstructions) to a computer processor. Examples of computer readablemedia include, but are not limited to, DVDs, CDs, hard disk drives,magnetic tape and servers for streaming media over networks.

As used herein, the terms “processor” and “central processing unit” or“CPU” are used interchangeably and refer to a device that is able toread a program from a computer memory (e.g., ROM or other computermemory) and perform a set of steps according to the program.

As used herein, the term “providing a prognosis” refers to providinginformation regarding the impact of the presence of CPPS (e.g., asdetermined by the diagnostic methods of the present invention) on asubject's future health (e.g., likelihood of responding to therapy).

As used herein, the term “subject diagnosed with a CPPS” refers to asubject who has been tested and found to have CPPS. The CPPS may bediagnosed using any suitable method, including but not limited to, thediagnostic methods of the present invention.

As used herein, the term “initial diagnosis” refers to results ofinitial CPPS diagnosis (e.g. the presence or absence of CPPS).

As used herein, the term “non-human animals” refers to all non-humananimals including, but are not limited to, vertebrates such as rodents,non-human primates, ovines, bovines, ruminants, lagomorphs, porcines,caprines, equines, canines, felines, ayes, etc.

As used herein, the term “gene transfer system” refers to any means ofdelivering a composition comprising a nucleic acid sequence to a cell ortissue. For example, gene transfer systems include, but are not limitedto, vectors (e.g., retroviral, adenoviral, adeno-associated viral, andother nucleic acid-based delivery systems), microinjection of nakednucleic acid, polymer-based delivery systems (e.g., liposome-based andmetallic particle-based systems), biolistic injection, and the like. Asused herein, the term “viral gene transfer system” refers to genetransfer systems comprising viral elements (e.g., intact viruses,modified viruses and viral components such as nucleic acids or proteins)to facilitate delivery of the sample to a desired cell or tissue. Asused herein, the term “adenovirus gene transfer system” refers to genetransfer systems comprising intact or altered viruses belonging to thefamily Adenoviridae.

As used herein, the term “nucleic acid molecule” refers to any nucleicacid containing molecule, including but not limited to, DNA or RNA. Theterm encompasses sequences that include any of the known base analogs ofDNA and RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N-6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of apolypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region of a structural gene and thesequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb or more on either end such that thegene corresponds to the length of the full-length mRNA. Sequenceslocated 5′ of the coding region and present on the mRNA are referred toas 5′ non-translated sequences. Sequences located 3′ or downstream ofthe coding region and present on the mRNA are referred to as 3′non-translated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

As used herein, the term “heterologous gene” refers to a gene that isnot in its natural environment. For example, a heterologous geneincludes a gene from one species introduced into another species. Aheterologous gene also includes a gene native to an organism that hasbeen altered in some way (e.g., mutated, added in multiple copies,linked to non-native regulatory sequences, etc). Heterologous genes aredistinguished from endogenous genes in that the heterologous genesequences are typically joined to DNA sequences that are not foundnaturally associated with the gene sequences in the chromosome or areassociated with portions of the chromosome not found in nature (e.g.,genes expressed in loci where the gene is not normally expressed).

As used herein, the term “gene expression” refers to the process ofconverting genetic information encoded in a gene into RNA (e.g., mRNA,rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e., via theenzymatic action of an RNA polymerase), and for protein encoding genes,into protein through “translation” of mRNA. Gene expression can beregulated at many stages in the process. “Up-regulation” or “activation”refers to regulation that increases the production of gene expressionproducts (i.e., RNA or protein), while “down-regulation” or “repression”refers to regulation that decrease production. Molecules (e.g.,transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequencesthat are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region may contain sequencesthat direct the termination of transcription, posttranscriptionalcleavage and polyadenylation.

The term “wild-type” refers to a gene or gene product isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product that displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics (includingaltered nucleic acid sequences) when compared to the wild-type gene orgene product.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

As used herein, the terms “an oligonucleotide having a nucleotidesequence encoding a gene” and “polynucleotide having a nucleotidesequence encoding a gene,” means a nucleic acid sequence comprising thecoding region of a gene or in other words the nucleic acid sequence thatencodes a gene product. The coding region may be present in a cDNA,genomic DNA or RNA form. When present in a DNA form, the oligonucleotideor polynucleotide may be single-stranded (i.e., the sense strand) ordouble-stranded. Suitable control elements such as enhancers/promoters,splice junctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the expressionvectors of the present invention may contain endogenousenhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecomponent or contaminant with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is such present in a form orsetting that is different from that in which it is found in nature. Incontrast, non-isolated nucleic acids as nucleic acids such as DNA andRNA found in the state they exist in nature. For example, a given DNAsequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding a given protein includes, by way ofexample, such nucleic acid in cells ordinarily expressing the givenprotein where the nucleic acid is in a chromosomal location differentfrom that of natural cells, or is otherwise flanked by a differentnucleic acid sequence than that found in nature. The isolated nucleicacid, oligonucleotide, or polynucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acid,oligonucleotide or polynucleotide is to be utilized to express aprotein, the oligonucleotide or polynucleotide will contain at a minimumthe sense or coding strand (i.e., the oligonucleotide or polynucleotidemay be single-stranded), but may contain both the sense and anti-sensestrands (i.e., the oligonucleotide or polynucleotide may bedouble-stranded).

As used herein, the term “purified” or “to purify” refers to the removalof components (e.g., contaminants) from a sample. For example,antibodies are purified by removal of contaminating non-immunoglobulinproteins; they are also purified by the removal of immunoglobulin thatdoes not bind to the target molecule. The removal of non-immunoglobulinproteins and/or the removal of immunoglobulins that do not bind to thetarget molecule results in an increase in the percent of target-reactiveimmunoglobulins in the sample. In another example, recombinantpolypeptides are expressed in bacterial host cells and the polypeptidesare purified by the removal of host cell proteins; the percent ofrecombinant polypeptides is thereby increased in the sample.

As used herein, the term “co-administration” refers to theadministration of two or more compositions to a subject. Thecompositions may be administered to treat the same or differentdiseases, disorders, and/or conditions. The compositions may beadministered by the same route or different routes of administration(e.g. oral, parenateral, topical, intervenous, transmucosal, and/orinhalation routes). The compositions may be administered simultaneouslyor at different times. The compositions may be administeredsimultaneously, but formulated for release at different times or indifferent regions of the subject. The compositions may target the sameor different pathways within the subject.

DETAILED DESCRIPTION OF THE INVENTION

Prostatitis is the most frequent urologic diagnosis in men under the ageof 50, accounting for 8% of all office visits to urologists. Themajority of prostatitis cases are classified as chronic pelvic painsyndrome (CPPS, NIH Category III). It is subclassified into categoriesIIIA (inflammatory) and IIIB (non-inflammatory). While variousmechanisms including immunologic dysfunction, infectious, and neurologicdysfunction have been cited in the development of the syndrome, theetiology and pathogenesis of CPPS is poorly understood. Effectivetreatment for the CPPS remains uncertain. Factors complicating themanagement of this condition include its multifactorial pathogenesis,lack of a gold standard for diagnostic testing, and the methodologiclimitations of many treatment studies.

There is a substantial body of evidence that demonstrates the occurrenceof immunological activity within the prostate gland. However, the natureand cause of this activity, and whether it is detrimental to the host,has not been determined. Inflammatory infiltrates in the prostate arevery common. In one study of 162 cases of surgically resected prostatictissue, 98% possessed inflammatory infiltrates (Kohnen et al., J.Urology 121:755-60 (1979)). The infiltrating cells consisted ofmonocytes and activated T and B lymphocytes (Theyer et al., Lab Invest.66:96-107 (1992); Steiner et al., J. Urology 151:480-84 (1994)).

A rare form of prostatic inflammation, granulomatous prostatitis, hasbeen characterized, although the etiology of the inflammation is alsounknown. One major theory about the disease, however, is that itrepresents an immune reaction against self prostatic proteins induced byinfection or manipulation of the gland by previous biopsy or surgicalprocedure (Stillwell et al., J. Urology 138:320-23 (1987); Dhundee etal., Histopathology 18:435-41 (1991)).

The disease is also observed after instillation of BacillusCalmette-Guerin (BCG) into the bladder as a treatment for superficialbladder cancer (Bahnson, J. Urology 146:1368-69 (1991)).

Recent observations about the existence of subsets of CD4+ T cells hasyielded fundamental information about immune responses in humans. CD4+ Tcells can be separated into subsets based upon the patterns of cytokinesthey secrete (Mosmann, Ann NY Acad. Sci. 664:89-92 (1992)). CD4+ T cellsthat secrete, among other cytokines, IFN-γ and IL-2 are called T helper1 (Th1) cells. Th1 cells mediate cellular immunity, such as delayedhypersensitivity responses. CD4+ T cells that secrete, among othercytokines, IL-4 and IL-10 are termed T helper 2 (Th2). Th2 cells areassociated with antibody production and allergy. Immune responsesmediated by Th1 and Th2 cells can be characterized by the local cytokineenvironment during an immune response.

Zisman et al. found IgG anti-PSA antibody titers to be higher in theserum of men with benign prostate hyperplasia (BPH) compared to controls(Zisman et al., J. Urology 154:1052-55 (1995)). However, of 17 men withchronic prostatitis, no discernable difference was found in meanantibody titer as compared to controls. Zisman et al. speculate that animmunologic mechanism may play a role in the symptomatology of BPH. Analternative explanation is that a Th1 type of response may be occurringin patients with chronic prostatitis/chronic pelvic pain syndrome. Inthis event, no antibody response would be expected.

Nonbacterial prostatitis, recently defined as chronic pelvic painsyndrome (CPPS), is characterized by pelvic or perineal pain and isassociated with prostatic inflammation (Chronic Prostatitis Workshop,National Institute of Health, Bethesda, Md., Dec. 7-8, 1995). CPPS isthe most common urologic diagnosis in men less than 50 years of age, yetlittle is known about its etiology and treatment. Nonbacterialprostatitis has been traditionally defined by the identification ofwhite blood cells (WBC) in expressed prostatic secretions (EPS) in theabsence of bacterial infection (Meares et al., Invest Urol, 5: 492(1968)). Thus, the identification of inflammatory mediators such ascytokines in EPS are contemplated to be useful in the classification ofmen with CPPS.

Cytokines are small protein molecules produced and used by immune andinflammatory cells to communicate, control the environment, and regulatelocal and systemic events of the immune response. Most cytokines areproduced and released locally and mediate their effects at the site ofinjury, infection or inflammation by autocrine and paracrine mechanisms.A number of cytokines regulate inflammation, including interleukin-1(IL-1) and tumor necrosis factor alpha (TNF-α). IL-1α and IL-1β,collectively termed IL-1, share only 26% homology but act via the samehigh affinity receptor (Dower et al., J Exp Med, 162: 501 (1985)). IL-1has a wide range of target cells and acts to promote antigen specificimmune responses, inflammation and tissue repair. TNF-α is synthesizedby cells of the monocyte/macrophage lineage. Synthesis of TNF-α isinduced by bacterial proteins, viruses and fungal antigens, making itsrole in infection and inflammation prominent. To better understand thenature of CPPS, the inventors evaluated the EPS of men with no urologicdisease, Benign Prostatic Hyperplasia (BPH), CPPS and asymptomaticinflammatory prostatitis (AIP) for the presence of leukocytes andcytokines.

Previous studies by the inventors demonstrated that the cytokinesinterleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), IL-8, andepithelial neutrophil activating peptide-78 were significantly higher insamples from men with IIIa, but not IIIb, CPPS compared to controls(See, e.g., Nadler et al., Journal of Urology, 164: 214-218 (2000)).

The present invention provides compositions and methods for prostaticdisease (e.g., CPPS) diagnostics, including but not limited to, CPPSmarkers (e.g., mast cell markers). Accordingly, the present inventionprovides methods of characterizing samples (e.g., expressed prostaticsecretions or semen), kits for the detection of markers, as well as drugscreening and therapeutic applications. Further, the present inventionprovides compositions and methods for treatment and/or prevention ofCPPS.

I. Detection/Diagnosis/Characterization

The present invention provides markers whose expression is specificallyaltered in prostatic disease. Such markers find use in the diagnosis,detection, and/or characterization of prostatic disease (e.g., CPPS).

A. Identification of Markers

Experiments conducted during development of embodiments of the presentinvention demonstrate that mast cell tryptase is present, andsignificantly elevated, in samples of men with CPPS.

Thus, in some embodiments, the present invention provides biologicmarkers for CPPS (e.g., markers that identify mast cells). In someembodiments, detecting the levels of mast cell markers in a samplepermits diagnosis of or increased likelihood of prostatic disease (e.g.,CPPS). In some embodiments, biomarkers of the present invention (e.g.,mast cell tryptase) are used in order to further understand andcharacterize the etiology and pathogenesis of CPPS.

In some embodiments, presence of or levels of mast cell tryptase isdetected in a sample. In some embodiments, samples are obtained from asubject (e.g., a patient), and include, but are not limited to, fluids,solids, tissues, and gases. In some embodiments, biological samplesinclude, among other things, body fluids (e.g., semen or saliva), bloodproducts (e.g., plasma, serum and the like), and their component parts.In preferred embodiments, samples include expressed prostaticsecretions, seminal plasma and/or seminal fluid.

In some embodiments, the present invention provides detection of and/ormeasurement of mast cell tryptase as indicative of the presence orabsence of prostate disease (e.g., CPPS) in a subject. In someembodiments, the present invention provides diagnosis, characterization(e.g. identification of subtype), identification, etc. of CPPS based ondetection of mast cell tryptase. In some embodiments, the presentinvention provides diagnosis, characterization (e.g. identification ofsubtype), identification, etc. of CPPS based on detection of mast celltryptase above a threshold level (e.g. above a defined concentration ina body fluid). In some embodiments, patients are categorized (e.g., asIIIa and IIIb) and therapies or other interventions selected accordingto the levels of mast cell tryptase detected. It is contemplated that,in some embodiments, subjects with certain levels of mast cell tryptase(e.g., elevated levels), as compared to controls, are classified ashaving prostatic disease (e.g., CPPS).

In some embodiments, mast cell tryptase is detected in conjunction withother biomarkers (e.g. chemokines (e.g., MIP-1α and MCP-1)). In someembodiments, mast cell tryptase is detected individually (e.g. withoutother biomarkers). It is contemplated that, according to experimentsconducted during the development of the present invention, thatdetection of mast cell tryptase enhances the sensitivity of predictingdisease (e.g., prostatic) and classifying disease (e.g., CPPS) overother methods known to those in the art. In some embodiments, detectionof mast cell tryptase, is used in combination with detection of othermarkers (e.g. chemokines/cytokines), including, but not limited toMIP-1α, MCP-1, GMCSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, α-inteferon,γ-interferon, GRO-α, RENTES, fractalkine, VEGF, and TNF, in order todetect or classify disease (e.g., CPPS and CPSI, respectively). In someembodiments, detection of mast cell tryptase, is used in combinationwith symptom identification to detect or classify disease (e.g., CPPSand CPSI, etc.).

B. Detection of Markers

In some embodiments, the present invention provides methods fordetection of expression of prostatic disease (e.g., CPPS markers). Inpreferred embodiments, expression is measured directly (e.g., at the RNAor protein level). In some embodiments, expression is detected insamples (e.g., semen, seminal fluid, seminal plasma or expressedprostatic secretions). In other embodiments, expression is detected inbodily fluids (e.g., including but not limited to, plasma, serum, wholeblood, mucus, and urine). The present invention further provides panelsand kits for the detection of markers. In preferred embodiments, thepresence of a CPPS marker is used to provide a prognosis to a subject.For example, the detection of mast cell tryptase in samples isindicative of CPPS. The information provided is also used to direct thecourse of treatment. For example, if a subject is found to have a marker(e.g., as described herein) indicative of the presence of CPPS,therapies can be started immediately in place of or in addition to othertreatments that would have otherwise been used. In addition, if asubject is found to have a CPPS that is not responsive to othertherapies, the expense and inconvenience of such therapies can beavoided.

The present invention is not limited mast cell tryptase as a marker. Anysuitable marker that correlates with CPPS may be utilized, including butnot limited to, MIP-1α, MCP-1, GMCSF, IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,α-interferon, γ-interferon, GRO-α, RENTES, fractalkine, VEGF, and TNF.Additional markers are also contemplated to be within the scope of thepresent invention. Any suitable method may be utilized to identify andcharacterize markers suitable for use in the methods of the presentinvention. For example, in some embodiments, markers identified as beingup or down-regulated in CPPS, e.g. using gene expression microarraymethods, are further characterized using tissue microarray,immunohistochemistry, Northern blot analysis, siRNA or antisense RNAinhibition, mutation analysis, investigation of expression with clinicaloutcome, etc.

In some embodiments, the present invention provides a panel for theanalysis of a plurality of markers. The panel allows for thesimultaneous analysis of multiple markers correlating with CPPS. Forexample, a panel may include markers identified as correlating with CPPSCategories I-IV. Depending on the subject, panels may be analyzed aloneor in combination in order to provide the best possible diagnosis andprognosis. Markers for inclusion on a panel are selected by screeningfor their predictive value using any suitable method, including but notlimited to, those described in the illustrative examples below.

In other embodiments, the present invention provides an expressionprofile map comprising expression profiles of CPPS. Such maps can beused for comparison with patient samples. Comparisons can be madeutilizing any suitable method, including but not limited to, computercomparison of digitized data. The comparison data is used to providediagnoses and/or prognoses to patients.

1. Detection of RNA

In some preferred embodiments, CPPS markers (e.g., including but notlimited to, those disclosed herein) are detected by measuring theexpression of corresponding mRNA in a sample. mRNA expression may bemeasured by any suitable method (hybridization, amplification, mass,sequencing, etc.).

2. Detection of Protein

In other embodiments, gene expression of CPPS markers is detected bymeasuring the expression of the corresponding protein or polypeptide.Protein expression may be detected by any suitable method. In someembodiments, proteins are detected by immunohistochemistry or ELISA. Insome embodiments, proteins are detected by their binding to an antibodyraised against the protein.

3. Data Analysis

In some embodiments, a computer-based analysis program is used totranslate the raw data generated by the detection assay (e.g., thepresence, absence, or amount of a given marker or markers) into data ofpredictive value for a clinician. The clinician can access thepredictive data using any suitable means. Thus, in some preferredembodiments, the present invention provides the further benefit that theclinician, who is not likely to be trained in genetics or molecularbiology, need not understand the raw data. The data is presenteddirectly to the clinician in its most useful form. The clinician is thenable to immediately utilize the information in order to optimize thecare of the subject.

The present invention contemplates any method capable of receiving,processing, and transmitting the information to and from laboratoriesconducting the assays, information provides, medical personal, andsubjects. For example, in some embodiments of the present invention, asample (e.g., a semen, serum or urine sample) is obtained from a subjectand submitted to a profiling service (e.g., clinical lab at a medicalfacility, genomic profiling business, etc.), located in any part of theworld (e.g., in a country different than the country where the subjectresides or where the information is ultimately used) to generate rawdata. Where the sample comprises a tissue or other biological sample,the subject may visit a medical center to have the sample obtained andsent to the profiling center, or subjects may collect the samplethemselves (e.g., a urine or semen sample) and directly send it to aprofiling center. Where the sample comprises previously determinedbiological information, the information may be directly sent to theprofiling service by the subject (e.g., an information card containingthe information may be scanned by a computer and the data transmitted toa computer of the profiling center using an electronic communicationsystems). Once received by the profiling service, the sample isprocessed and a profile is produced (i.e., expression data), specificfor the diagnostic or prognostic information desired for the subject.

The profile data is then prepared in a format suitable forinterpretation by a treating clinician. For example, rather thanproviding raw expression data, the prepared format may represent adiagnosis or risk assessment (e.g., Category III CPPS) for the subject,along with recommendations for particular treatment options. The datamay be displayed to the clinician by any suitable method. For example,in some embodiments, the profiling service generates a report that canbe printed for the clinician (e.g., at the point of care) or displayedto the clinician on a computer monitor.

In some embodiments, the information is first analyzed at the point ofcare or at a regional facility. The raw data is then sent to a centralprocessing facility for further analysis and/or to convert the raw datato information useful for a clinician or patient. The central processingfacility provides the advantage of privacy (all data is stored in acentral facility with uniform security protocols), speed, and uniformityof data analysis. The central processing facility can then control thefate of the data following treatment of the subject. For example, usingan electronic communication system, the central facility can providedata to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the datausing the electronic communication system. The subject may chose furtherintervention or counseling based on the results. In some embodiments,the data is used for research use. For example, the data may be used tofurther optimize the inclusion or elimination of markers as usefulindicators of a particular condition associated with the disease.

4. Kits

In yet other embodiments, the present invention provides kits for thedetection and characterization of prostatic disease. In someembodiments, the kits contain antibodies specific for a CPPS marker, inaddition to detection reagents and buffers. In other embodiments, thekits contain reagents specific for the detection of mRNA or cDNA (e.g.,oligonucleotide probes or primers). In preferred embodiments, the kitscontain all of the components necessary, sufficient, or useful toperform a detection assay, including all controls, directions forperforming assays, and any necessary software for analysis andpresentation of results.

II. Treatment/Prevention

In some embodiments, the present invention provides compositions andmethods for inhibition of mast cell function to treat or prevent CPPS.In some embodiments, the present invention provides compositions andmethods for inhibition of CPPS markers to treat or prevent chronicpelvic pain syndrome. In some embodiments, the present inventionprovides compositions and methods for inhibition of mast cell markers totreat or prevent chronic pelvic pain syndrome. In some embodiments, thepresent invention provides compositions and methods for inhibition ofMCP-1 and/or MIP-1α to treat or prevent chronic pelvic pain syndrome. Insome embodiments, the present invention provides compositions andmethods for inhibition of MCP-1 to treat or prevent chronic pelvic painsyndrome. In some embodiments, the present invention providescompositions and methods for inhibition of MIP-1α to treat or preventchronic pelvic pain syndrome. In some embodiments, chronic pelvic painsyndrome is treated or prevented by inhibition of mast cell function andone or more additional therapies (e.g. inhibition of MCP-1 and MIP-1α).In some embodiments, the present invention provides co-administration ofone or more inhibitors of mast cell function and one or more inhibitorsof MCP-1 and/or MIP-1α.

In some embodiments, the present invention provides a method oftreating, preventing, or ameliorating signs or symptoms of CPPS and/orrelated or similar diseases, conditions, or disorders in a subject. Insome embodiments, compositions and methods of the present invention areprovided prophylactically (e.g. to prevent development of CPPS orsymptoms thereof). In some embodiments, compositions and methods of thepresent invention are provided therapeutically (e.g. to treat CPPS orsymptoms thereof in a subject suffering from CPPS). In some embodiments,compositions and methods of the present invention provide palliativetreatment (e.g. reduction in the symptoms of CPPS). In some embodiments,compositions and methods of the present invention provide curativetreatment (e.g. elimination of CPPS in a subject). In some embodiments,compositions and methods of the present invention provide preventativetreatment (e.g. prevent the development of CPPS in a subject).

In some embodiments, the present invention provides compositions andmethods to treat or prevent CPPS in a subject (e.g. mammal, canine,rodent, primate, human, etc.) via co-administration of one or moreinhibitors of mast cell function (e.g. mast cell stabilizer, histaminereceptor antagonist, etc.), an inhibitor of one or more CPPS markers(e.g. inhibitor of MCP-1, inhibitor of MIP-1α, etc.). In someembodiments, the present invention provides compositions and methods totreat or prevent CPPS in a subject via co-administration of two or moreof: an inhibitor of mast cell function (e.g. mast cell stabilizer,histamine receptor antagonist, etc.), an inhibitor of MCP-1, and aninhibitor of MIP-1α. In some embodiments, the present invention providesone or more of each of: a mast cell stabilizer, a histamine receptorantagonist, an inhibitor of MCP-1, and an inhibitor of MIP-1α. In someembodiments, the present invention provides pharmaceutical compositionscomprising a therapeutically effective amount of one or more inhibitorsof mast cell function, one or more inhibitors of MCP-1, one or moreinhibitors of MIP-1α, and optionally, appropriate pharmaceuticallyacceptable carriers. In some embodiments, the present invention providespharmaceutical compositions comprising one or more of a mast cellstabilizer, a histamine receptor antagonist, an inhibitor of MCP-1,and/or an inhibitor of MIP-1α. In some embodiment, the present inventionprovides co-administration of one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8,9, 10, etc.) inhibitors of mast cell function, inhibitors of MCP-1,and/or inhibitors of MIP-1α.

In some embodiments, the present invention provides one or moreinhibitors of mast cell function, and methods of use thereof. In someembodiments, an inhibitor of mast cell function inhibits mast celldegranulation. In some embodiments, an inhibitor of mast cell functionis a mast cell stabilizer (e.g. cromolyn sodium). In some embodiments,an inhibitor of mast cell function inhibits the release of histaminefrom mast cells. In some embodiments, an inhibitor of mast cell functionis an inhibitor of a histamine receptor (e.g. histamine receptor 1antagonist (e.g. cetirizine), histamine receptor 2 antagonist (e.g.ranitidine)). In some embodiments, the present invention providesadministration of one or more mast cell stabilizers (e.g. cromolynsodium) and one or more histamine receptor antagonists (e.g. histaminereceptor 1 antagonist (e.g. cetirizine), histamine receptor 2 antagonist(e.g. ranitidine)). In some embodiments, the present invention providesadministration of one or more mast cell stabilizer (e.g. cromolynsodium), histamine receptor 1 antagonist (e.g. cetirizine), and/orhistamine receptor 2 antagonist (e.g. ranitidine)). In some embodiments,suitable mast cell stabilizers (e.g. inhibitors of degranulation)include, but are not limited to cromolyn sodium, lodoxamide, nedocromil,and derivatives thereof. In some embodiments, suitable histaminereceptor 1 antagonists include, but are not limited to: clemastine,diphenhydramine, doxylamine, loratadine, desloratadine, fexofenadine,pheniramine, cetirizine, ebastine, promethazine, chlorpheniramine,levocetirizine, quetiapine, meclizine, dimenhydrinate, and derivativesthereof. In some embodiments, suitable histamine receptor 2 antagonistsinclude, but are not limited to: cimetidine, famotidine, ranitidine,nizatidine, roxatidine, lafutidine, and derivatives thereof.

Where one or more therapeutic agents are to be administered to a subject(e.g., a mast cell stabilizer and a histamine receptor antagonist), theagent may be formulated together and administered simultaneously or maybe formulated separately and administered simultaneously or in sequencevia any desired timing.

In some embodiments, the present invention provides one or moreinhibitors of biomarkers of CPPS (e.g. including but not limited to,MCP-1, MIP-1α, and mast cell tryptase). In some embodiments, the presentinvention provides one or more inhibitors of MCP-1, MIP-1α, and/or mastcell tryptase. In some embodiments, the present invention provides oneor more inhibitors of MCP-1 and/or MIP-1α. In some embodiments,therapies target CPPS markers (e.g., including but not limited to,MCP-1, MIP-1α, mast cell markers (e.g. mast cell tryptase)). Suitablecompositions and method of inhibiting CPPS markers (e.g. MCP-1 and/orMIP-1α) are provided in U.S. Pat. App. No. 20050277140, hereinincorporated by reference in its entirety. In some embodiments,inhibitors of biomarkers of CPPS (e.g. MCP-1 and/or MIP-1α) include, butare not limited to antisense oligonucleotides, genetic therapies,anti-chemokine therapy, etc.

In some embodiments, the present invention targets the expression ofCPPS markers (e.g. by antisense therapy). For example, in someembodiments, the present invention employs compositions comprisingoligomeric antisense compounds (e.g., siRNA, miRNA, anti-senseoligonucleotides), particularly oligonucleotides (e.g., those identifiedin the drug screening methods described above), for use in modulatingthe function of nucleic acid molecules encoding CPPS markers of thepresent invention, ultimately modulating the amount of CPPS markerexpressed. This is accomplished by providing antisense compounds thatspecifically hybridize with one or more nucleic acids encoding CPPSmarkers and interfere with the normal function of the nucleic acid. Insome embodiments, expression of CPPS markers is inhibited to treatand/or prevent CPPS and/or symptoms associate therewith. The presentinvention also includes pharmaceutical compositions and formulationsthat include the antisense compounds of the present invention asdescribed below.

In some embodiments, the present invention contemplates the use of anygenetic manipulation for use in modulating the expression of CPPSmarkers of the present invention. Examples of genetic manipulationinclude, but are not limited to, gene knockout (e.g., removing the CPPSmarker gene from the chromosome using, for example, recombination),expression of antisense constructs with or without inducible promoters,and the like. Delivery of nucleic acid construct to cells is achieved byany of various methods including, but not limited to, directed injectionof naked DNA constructs, bombardment with gold particles loaded withsaid constructs, and macromolecule mediated gene transfer using, forexample, liposomes, biopolymers, and the like.

In some embodiments, the present invention provides compounds whichinterfere with the production and/or activity of various cytokines (e.g.anti-cytokine compounds), by inhibiting production, processing oractivity of a cytokine or its receptor. Thus, the invention alsoprovides a method for treating subjects determined to be suffering fromCPPS or other disorders associated with elevated levels of one or morecytokines in one or more components or fractions of semen, preferablyseminal plasma, comprising administering one or more anti-cytokineagents, including, but not limited to those disclosed in U.S. Pat. No.6,180,355.

In some embodiments, the present invention provides treatment and/orprevention of CPPS through administration of two or more therapeuticcompounds (e.g. 1, 2, 3, 4, 5, 6 compounds). In some embodiments, thetherapeutic compounds are formulated together into a singlepharmaceutical composition (e.g. pill, topically-administered liquid,inhalant, etc.). In some embodiments, the therapeutic compoundsformulated together within a pharmaceutical composition are configuredfor separate therapeutic release regimens (e.g. timed release, delayedrelease, immediate release, etc.). In some embodiments, the therapeuticcompounds formulated together within a pharmaceutical composition areconfigured for immediate effectiveness. In some embodiments, thetherapeutic compounds are formulated as separate pharmaceuticalcompositions to be co-administered. In some embodiments,co-administration comprises administering separate pharmaceuticalcompositions simultaneously, or near simultaneously. In someembodiments, co-administration comprises a therapeutic strategy in whicha subject is administered separate pharmaceutical compositions, but notnecessarily together.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient. Theadministering physician can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models or based on the examples described herein. Ingeneral, dosage is from 0.01 μg to 100 g per kg of body weight, and maybe given once or more daily, weekly, monthly or yearly. The treatingphysician can estimate repetition rates for dosing based on measuredresidence times and concentrations of the drug in bodily fluids ortissues. Following successful treatment, it may be desirable to have thesubject undergo maintenance therapy to prevent the recurrence of thedisease state, wherein the oligonucleotide is administered inmaintenance doses, ranging from 0.01 μg to 100 g per kg of body weight,once or more daily, to once every 20 years.

III. Drug Screening

In some embodiments, the present invention provides drug screeningassays (e.g., to screen for CPPS drugs). The screening methods of thepresent invention utilize CPPS markers identified using the methods ofthe present invention (e.g., including but not limited to, mast cellmarkers). For example, in some embodiments, the present inventionprovides methods of screening for compound that directly or indirectlyalter (e.g., increase or decrease) the expression of CPPS marker genes.

In one screening method, candidate compounds are evaluated for theirability to alter CPPS marker expression by contacting a compound with acell expressing a CPPS marker and then assaying for the effect of thecandidate compounds on expression. In some embodiments, the effect ofcandidate compounds on expression of a CPPS marker gene is assayed forby detecting the level of CPPS marker mRNA expressed by the cell. mRNAexpression can be detected by any suitable method. In other embodiments,the effect of candidate compounds on expression of CPPS marker genes isassayed by measuring the level of polypeptide encoded by the CPPSmarkers. The level of polypeptide expressed can be measured using anysuitable method, including but not limited to, those disclosed herein.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1 Treating Chronic Pelvic Pain Syndrome

The experimental autoimmune prostatitis (EAP) model was selected forexperiments conducted during development of embodiments of the presentinvention. Autoimmunity is one of the etiologies for patients with CPPS.Rat prostate antigen instillation in NOD mice provokes a chronicinflammation that is multifocal and evenly distributed in all theprostatic lobes. Pelvic pain was examined in NOD and C57BL/6 (B6) miceusing quantitative behavior testing methods to assess tactilesensitivity of the suprapubic region. Mechanical stimulation of thepelvic area of sham-immunized mice with Von Frey fibers resulted in aresponse frequency that correlated with the applied force, and thisresponse profile did not change during the 30-day course of theexperiment (SEE FIG. 1). In contrast, EAP induced NOD mice exhibitedenhanced sensitivity to pelvic stimuli that was significantly greater atall filaments by post-immunization day 10 in NOD mice (SEE FIG. 1) andwas sustained through the end of the experiment on day 30. Whenexpressed as increase in pain from baseline there was an approximately350% increase in pain behavior compared to sham controls (SEE FIG. 1).This indicates the development of chronic pelvic pain in EAP mice and isthe first report of chronic pain symptoms in an animal model ofprostatitis.

Experiments were conducted during development of embodiments of thepresent invention to determine whether chronic pain development wasmouse strain specific by using wild-type C57BL/6 (B6) mice. InEAP-induced B6 mice, sensitivity was increased to mechanical stimulationbut to a lesser magnitude (SEE FIG. 2B). EAP induced no changes intactile sensitivity of the plantar region of the hind paw (SEE FIG. 2C).Therefore, EAP induces chronic pain specific to the pelvic region inboth NOD and B6 mice. To confirm that the effects of prostate antigen(PAg) were specific to pain behavior, normal behaviors during freeroaming were quantified in both mouse strains. PAg induced nosignificant differences in grooming, cage crossing, or rearing,suggesting that pelvic pain is evoked and not due to spontaneous pain.There was also no prolonged change in weight indicating that PAg is notassociated with dramatic changes in gross physiology. Thus, painspecific to the suprapubic area can be recreated in a murine modelthrough EAP and persists in a chronic fashion without any gross systemiceffects on the animal. The EAP model represents a chronic model whichtransitions from a focus on prostatitis etiology to one on the symptomsof the disease. The model closely reflects CPPS, a syndrome that hasuncertain etiology and is diagnosed based on symptoms.

Mast cell tryptase has been used in a variety of human diseaseconditions as a biomarker for total mast cell number and activation.Experiments conducted during development of embodiments of the presentinvention, using an autoimmune murine model that recapitulates aspectsof CPPS including the presence of chronic pelvic pain, demonstrated asignificant increase in total and activated mast cells in the prostatesof mice with pelvic pain. Prostates from mice were examined for totalmast cell numbers as well as activation status of the mast cells. Mastcells were classified as resting, partially activated or activateddepending on the dispersal of toluidine stained granules. Resting mastcells contained greater than 90% of visible granules in the cellboundary. Partially active mast cells showed approximately 10% to 20% ofvisible granules beyond the cell boundary. Activated mast cellsdemonstrated greater than 20% of visible granules beyond the cellboundary, while granule dispersion was typically greater than 50% inactive cells. Total mast cells were observed to be increased 5 daysafter induction of EAP with majority of cells in the resting stage. Byday 10 there was significant activation of mast cells that was notobserved at 20 and 30 days (SEE FIG. 3). However, the apparent reductionin resting cells at 20 and 30 days with the absence of any increase inactivated cells suggests that some late stage activated cells are notdetected by toluidine blue staining. Thus EAP causes significantincrease in mast cell numbers and results in mast cell activation attime points that also correspond to elevated pain symptoms.

The requirement for mast cell deficient mice to mediate pain in theanimal model was confirmed by examining the ability of mast celldeficient mice (Kit^(W-sh)/Kit^(W-sh)) to exhibit tactile allodynia ofthe pelvic region upon induction of autoimmune prostatitis. Wild-typeC57BL/6 mice developed robust pelvic pain behavior by day 5 afterantigen administration that persisted at day 10 (SEE FIG. 4A). Incontrast, Kit^(W-sh)/Kit^(W-sh) mice did not show any increase in pelvicpain behavior at 5 days and showed inhibited pain responses at 10 daysafter antigen administration (SEE FIG. 4B). In addition, both groups ofmice exhibited no changes in pain responses in the footpad orsignificant changes in body weight. These data indicate that mast cellsare involved in the development of pelvic pain behavior.

Experiments conducted during development of embodiments of the presentinvention demonstrated that mast cell deficient mice and wild-type micedevelop similar levels of EAP-induced chronic inflammation. The extentof chronic inflammation in mast cell deficient mice(Kit^(W-sh)/Kit^(W-sh)) was compared to wild-type C57BL/6 mice uponinduction of autoimmune prostatitis. Inflammation in both groups of micewas chronic with multiple foci of inflammatory cells distributedpredominantly in the periglandular regions and stroma of the prostate(SEE FIGS. 5A and 5B). Both groups of mice developed chronicinflammation that was not statistically different when quantified usingstandard inflammation scoring (SEE FIG. 5E). In contrast, toluidine bluestaining for mast cells revealed large numbers of mast cells in variousstages of activation in the stroma of C57BL/6 but notKit^(W-sh)/Kit^(W-sh) mice (see arrows SEE FIGS. 5C and 5D). Thus, mastcells are not a requirement for the development of inflammation, buthave a critical role for the establishment of chronic pain.

Experiments conducted during development of embodiments of the presentinvention demonstrated that anti-MCP-1 or anti-MIP-1α antibodies reducepelvic pain development. Experiments were conducted to examine whetheranti-MCP-1 therapy could be used to inhibit the development of pelvicpain in the animal model. Commercially derived (R&D systems) polyclonalanti-MCP-1, anti-MIP-1α and an isotype control antibody were used toexamine their ability to prevent the development of pelvic pain.Antibodies were administered to NOD mice at the time of initiation ofautoimmunity and pain was evaluated at 5, 10, 20 and 30 days postinjection. A significant decrease of 68% was observed in pain responsesusing the anti-MCP-1 (SEE FIG. 6B), 61% using the anti-MIP-1α (SEE FIG.6C) and no reduction in pain using the control antibody (SEE FIG. 7) at5, 10 and 20 days after antibody administration. Pain responses returnedto pre-antibody administration levels by 30 days suggesting that theinhibitory action of the antibody was no longer retained. These datademonstrate that pelvic pain in this model of CP/CPPS can be inhibitedusing antibodies targeting MCP-1 and MIP-1α. These results indicate thatanti-MCP-1 or anti-MIP-1α are therapeutic targets that reduce pelvicpain in CPPS. Therapeutic use of these antibodies to reduce pain afterit had developed in the EAP model was examined (SEE FIG. 7). Antibodieswere administered at 20 days after EAP induction based on our earlierresults indicating peak expression at 20 days. Anti-MCP-1 was able tosignificantly reduce pain for up to 2 days following treatment afterwhich time pain returned to control levels. Anti-MIP-1 alpha antibodiesshowed a reduction in pain symptoms that was, however, not statisticallysignificant. These results indicate that chemokine inhibition is aviable therapeutic mechanism to reduce pelvic pain symptoms.

Experiments conducted during development of embodiments of the presentinvention demonstrated that pharmacological inhibitors of mast cellfunction therapeutically reduce pelvic pain behavior in mice. Pelvicpain behavior in the EAP model was inhibited following the establishmentof pain. EAP was induced in NOD mice and pelvic pain behavior atbaseline and 10 days after disease onset was measured. Mice wereadministered a mast cell stabilizer, cromolyn sodium; a histaminereceptor 1 antagonist, cetirizine; a histamine receptor 2 antagonist,ranitidine; and a combination of all three pharmacological agents orsaline as control. Pelvic pain behavior was tested one hour after drugadministration. Results indicate that treatment with the combination ofall three pharmacological agents reduced pain behavior by 64% (SEE FIGS.8E and 8F) while treatment with cromolyn sodium alone reduced pain by39.6% (SEE FIGS. 8D and 8F). In contrast, treatment with H1 and H2receptor antagonists reduced pain behavior by 13.6% and 15.9%respectively while controls exhibited no reduction in pain behavior.These results clearly indicate that mast cells and their granulecontents play an important role in the continued presence of pelvic painin the EAP animal model. These data also indicate that therapeuticintervention in established chronic pelvic pain may be achieved byselective targeting of mechanisms of mast cell activation anddegranulation.

Experiments conducted during development of embodiments of the presentinvention demonstrate that pelvic pain behavior is inhibited bytargeting MCP-1 and MIP-1α, as well as inhibitors of mast celldegranulation and function. These studies indicate that therapeuticstrategies that are multimodal and inhibit multiple actors in thepathogenesis of pelvic pain are effective in the treatment of CPPS.

Example 2 Mast Cell Tryptase is a Biomarker for CPPS

Mast cell tryptase has been used in a variety of human diseaseconditions as a biomarker for total mast cell number and activation.Experiments conducted during development of embodiments of the presentinvention, using an autoimmune murine model that recapitulates aspectsof CPPS including the presence of chronic pelvic pain, demonstrated asignificant increase in total and activated mast cells in the prostatesof mice with pelvic pain. Prostates from mice were examined for totalmast cell numbers as well as activation status of the mast cells. Mastcells were classified as resting, partially activated or activateddepending on the dispersal of toluidine stained granules. Resting mastcells contained greater than 90% of visible granules in the cellboundary. Partially active mast cells showed approximately 10% to 20% ofvisible granules beyond the cell boundary. Activated mast cellsdemonstrated greater than 20% of visible granules beyond the cellboundary, while granule dispersion was typically greater than 50% inactive cells. Total mast cells were observed to be increased 5 daysafter induction of EAP with majority of cells in the resting stage. Byday 10 there was significant activation of mast cells that was notobserved at 20 and 30 days (SEE FIG. 3). However, the apparent reductionin resting cells at 20 and 30 days with the absence of any increase inactivated cells suggests that some late stage activated cells are notdetected by toluidine blue staining. Thus EAP causes significantincrease in mast cell numbers and results in mast cell activation attime points that also correspond to elevated pain symptoms.

The requirement for mast cell deficient mice to mediate pain in theanimal model was confirmed by examining the ability of mast celldeficient mice (Kit^(W-sh)/Kit^(W-sh)) to exhibit tactile allodynia ofthe pelvic region upon induction of autoimmune prostatitis. Wild-typeC57BL/6 mice developed robust pelvic pain behavior by day 5 afterantigen administration that persisted at day 10 (SEE FIG. 4A). Incontrast, Kit^(W-sh)/Kit^(W-sh) mice did not show any increase in pelvicpain behavior at 5 days and showed inhibited pain responses at 10 daysafter antigen administration (SEE FIG. 4B). In addition, both groups ofmice exhibited no changes in pain responses in the footpad orsignificant changes in body weight. These data indicate that mast cellsare involved in the development of pelvic pain behavior.

Experiments conducted during development of embodiments of the presentinvention using clinical samples of expressed prostatic fluid (EPS) frompatients with CPPS IIIb, benign prostatic hypertrophy (BPH), and controlsubjects demonstrate that mast cell tryptase provides a biomarker forCPPS (e.g. for diagnosis and/or characterization of CPPS). The presenceof mast cell tryptase was measured using a colorimetric substrate basedassay (Millipore) as an index of mast cell degranulation. EPS samplesfrom CPPS patients demonstrated a significant increase in tryptaselevels when compared to controls or BPH patient samples (SEE FIG. 3). Incontrast, BPH samples did not show a significant increase in tryptaseexpression from controls (SEE FIG. 3). These data indicate that mastcell tryptase provides a biomarker for identification, detection, and/orcharacterization of CPPS in humans. These data indicate that mast cellsand their degranulation products play a role in CPPS pathogenesis.

Example 3 Synergism, Dosing, and Administration

Experiments were conducted to: (1) identify synergism between a mastcell inhibitor (cromolyn sodium) and a histamine 1 receptor antagonist(cetirizine), (2) identify optimized dose of cromolyn sodium thatexhibits synergism with a fixed dose of cetirizine, and (3) identify theoptimized route of administration for the combination therapy (SEE FIG.11). The results indicated that the combination treatment of CS andCetrizine is significantly more effective than CS alone in reducingpelvic pain in EAP mice. Synergism is assumed when the dose ofcombination producing a 50% effect is less than the dose from the 50%effect produced by a purely additive mixture of the two components ofthe combination. Intraprostatic lidocaine was utilized as the standardfor maximal pain benefit. The 24 hour time point and the 1 hour timepoint after the second treatment are the time points in whichsignificant pain reduction was found for the oral therapy. Oral (SEEFIG. 12), intraperitoneal (SEE FIG. 13), and intravenous (SEE FIG. 14)administration was tested.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in therelevant fields are intended to be within the scope of the followingclaims.

We claim:
 1. A composition for the treatment of chronic pelvic painsyndrome (CPPS), comprising: a) a mast cell stabilizer; and b) ahistamine receptor 1 antagonist and/or a histamine receptor 2antagonist; wherein the composition is formulated for administration bya route other than inhalation.
 2. The composition of claim 1, furthercomprising an inhibitor of MIP-1α and/or MCP-1, wherein the inhibitor isan antibody which binds MCP-1 and/or MIP-1α, respectively.
 3. Thecomposition of claim 1, wherein the mast cell stabilizer is cromolynsodium.
 4. The composition of claim 1, wherein the histamine receptor 1antagonist is cetirizine.
 5. The composition of claim 1, wherein thehistamine receptor 2 antagonist is ranitidine.